Sauer:Lysing E. coli with Lysozymes

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(Hen Egg White Lysozyme)
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Submitted by [[User:Smoore|Sean Moore]]
Submitted by [[User:Smoore|Sean Moore]]
==Getting The Most Out Of Your Bugs==
==Getting The Most Out Of Your Bugs==
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Native lysis is a staple protocol in practically every biochemistry lab, yet there is significant variability in the procols I have read.  The intention is to liberate the guts of E. coli without disturbing the native conformations of the biomolecules.  Using a French pressure cell you can sheer the cells open, this is a great way to lyse cells.  Unfortunately, it is impractical for multiple samples or small-volume samples that don't fit in the pressure cell.  Besides, the machine is scary, lysis can be variable, and cleanup is a hassle.  Chickens and bacteriophage have evolved a great way of opening E. coli using enzymes.  Most commercial lysozymes are free from proteolytic activity and can be added in large amounts.  Be careful, if your protein is ~14 kDa you may inadvertantly purify the added lysozyme instead of your target protein.  Don't laugh, it's happened more than once and I have met people who solved the crystal stucture of lysozyme by mistake.
===Hen Egg White Lysozyme===
===Hen Egg White Lysozyme===
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I prepare the lysozyme fresh each time from lyophilized powder.  Pre-warm the bottle to prevent moisture from condensing in the bottle when it's opened.  Pure B-Per doesn't seem to allow it into solution and it remains mostly as an inactive precipitate. So I make a ~10 mg/mL stock in some buffer off my shelf (I usually use 50 mM bis-Tris, pH 6.5).  Then I dilute from this to 0.1 to 0.01 mg/mL in the B-Per lysis solution.  To save time, I use a pipette tip and lift out dried lysozyme horizontally from the bottle, I then place a microfuge tube over the tip and shake off the powder.  I measured the before and after weight of several tubes and found that I can make pretty close to a 10 mg/mL solution by just adding 400 uL of buffer to the tube.  So, I no longer weigh out lysozyme each time, I just assume that my tip carries about 4 mg of protein.
+
I prepare the lysozyme fresh each time from lyophilized powder.  Pre-warm the bottle to prevent moisture from condensing in the bottle when it's opened.  Pure B-Per and most lysis buffers don't seem to allow it into solution well and it remains mostly as an inactive precipitate. So I make a ~10 mg/mL stock in some buffer off my shelf (I usually use 50 mM bis-Tris, pH 6.5).  Then I dilute from this to 0.1 to 0.01 mg/mL in the B-Per lysis solution.  To save time, I use a 200 μL pipette tip and lift out dried lysozyme horizontally from the bottle, I then place a microfuge tube over the tip and shake off the powder.  I measured the before and after weight of several tubes and found that I can make pretty close to a 10 mg/mL solution by just adding 400 μL of buffer to the tube.  So, I no longer weigh out lysozyme each time, I just assume that my tip carries about 4 mg of protein.
 +
 
 +
In most cases, simply adding lysozyme to your resuspended cells and waiting is sufficient to invoke lysis.  Some protocols call for placing the cells on ice during lysis in an effort to curb proteolytic activity.  This never made much sense to me,  you also slow down the activity of the lysozyme.  I have never seen a comparison of the activities of proteases and lysozyme at 0 and 25 degrees, but I generally lyse at room temperature.
 +
 
 +
Lysis is apparent by a reduction in turbidity and a severe increase in viscosity from the liberated chromosomes.  For protein preparations, I usually add the nuclease Benzonase to reduce the viscosity because it has activity toward double- and single-stranded DNA and RNA.  If you just add DNase I, be aware that it is frequently "purified" from pancreas and can be loaded with proteases.  If you see a lot of damage to your protein during lysis, add protease inhibitiors and/or change nuclease.  Additionally, DNase I requires both magnesium and calcium for activity so make sure your buffer has these.
===Intracellular Lysozymes===
===Intracellular Lysozymes===
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One problem with egg lysozyme is that it doesn't seem to work well at high Mg++ concentrations, in certain buffers, or in cells that are in stationary phase.  So, if you want to make a lysate in a defined buffer (like ribosomes in 10 mM Mg++), lysis is greatly impeded.  To fix this, I made a new "lysis plasmid" inspired by the pLysS/E plasmids sold by Novagen.  pLysS constitutively expresses the T4 lysozyme.  It is intended to inhibit the activity of T4 polymerase in T4 expression systems.  It has the added benefit that disruption of the inner membrane allows the lysozyme to get to the cell wall and lyse the cells.  So, freezing and thawing without a cryoprotectant or adding chloroform to the cells causes lysis.  The problem with pLysS is that cells harboring it are sick and lyse during centrifugation, the lysozyme is not a dedicated lysozyme (it has other cellular functions), and that it doesn't seem to lyse stationary phase cells well.  So, after speaking with Ryland Young, I decided to make a better plasmid.
 +
 +
In my construct, I placed the phage Lambda lysozyme "R gene" under constitutive control of the moderate Bla promoter from beta-lactamase.  This is a promoter that appears to exhibit even expression throughout growth (reference coming, some RNA micro-array paper).  Cells harboring this plasmid grow well and can be frozen if there is at least 10% glycerol present.  To lyse these cells, I add 10 uL of chloroform per mL of liquid and vortex.  At room temp, the cells completely lyse in about a minute.  Cells harboring the lysis plasmid can be transformed with chemical methods (CsCl, TSS, etc.) but NOT by electroporation.
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[[Category:Protocol]]
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[[Category:In vitro]]
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[[Category:Escherichia coli]]
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[[Category:Protein]]

Current revision

Submitted by Sean Moore

Getting The Most Out Of Your Bugs

Native lysis is a staple protocol in practically every biochemistry lab, yet there is significant variability in the procols I have read. The intention is to liberate the guts of E. coli without disturbing the native conformations of the biomolecules. Using a French pressure cell you can sheer the cells open, this is a great way to lyse cells. Unfortunately, it is impractical for multiple samples or small-volume samples that don't fit in the pressure cell. Besides, the machine is scary, lysis can be variable, and cleanup is a hassle. Chickens and bacteriophage have evolved a great way of opening E. coli using enzymes. Most commercial lysozymes are free from proteolytic activity and can be added in large amounts. Be careful, if your protein is ~14 kDa you may inadvertantly purify the added lysozyme instead of your target protein. Don't laugh, it's happened more than once and I have met people who solved the crystal stucture of lysozyme by mistake.

Hen Egg White Lysozyme

I prepare the lysozyme fresh each time from lyophilized powder. Pre-warm the bottle to prevent moisture from condensing in the bottle when it's opened. Pure B-Per and most lysis buffers don't seem to allow it into solution well and it remains mostly as an inactive precipitate. So I make a ~10 mg/mL stock in some buffer off my shelf (I usually use 50 mM bis-Tris, pH 6.5). Then I dilute from this to 0.1 to 0.01 mg/mL in the B-Per lysis solution. To save time, I use a 200 μL pipette tip and lift out dried lysozyme horizontally from the bottle, I then place a microfuge tube over the tip and shake off the powder. I measured the before and after weight of several tubes and found that I can make pretty close to a 10 mg/mL solution by just adding 400 μL of buffer to the tube. So, I no longer weigh out lysozyme each time, I just assume that my tip carries about 4 mg of protein.

In most cases, simply adding lysozyme to your resuspended cells and waiting is sufficient to invoke lysis. Some protocols call for placing the cells on ice during lysis in an effort to curb proteolytic activity. This never made much sense to me, you also slow down the activity of the lysozyme. I have never seen a comparison of the activities of proteases and lysozyme at 0 and 25 degrees, but I generally lyse at room temperature.

Lysis is apparent by a reduction in turbidity and a severe increase in viscosity from the liberated chromosomes. For protein preparations, I usually add the nuclease Benzonase to reduce the viscosity because it has activity toward double- and single-stranded DNA and RNA. If you just add DNase I, be aware that it is frequently "purified" from pancreas and can be loaded with proteases. If you see a lot of damage to your protein during lysis, add protease inhibitiors and/or change nuclease. Additionally, DNase I requires both magnesium and calcium for activity so make sure your buffer has these.

Intracellular Lysozymes

One problem with egg lysozyme is that it doesn't seem to work well at high Mg++ concentrations, in certain buffers, or in cells that are in stationary phase. So, if you want to make a lysate in a defined buffer (like ribosomes in 10 mM Mg++), lysis is greatly impeded. To fix this, I made a new "lysis plasmid" inspired by the pLysS/E plasmids sold by Novagen. pLysS constitutively expresses the T4 lysozyme. It is intended to inhibit the activity of T4 polymerase in T4 expression systems. It has the added benefit that disruption of the inner membrane allows the lysozyme to get to the cell wall and lyse the cells. So, freezing and thawing without a cryoprotectant or adding chloroform to the cells causes lysis. The problem with pLysS is that cells harboring it are sick and lyse during centrifugation, the lysozyme is not a dedicated lysozyme (it has other cellular functions), and that it doesn't seem to lyse stationary phase cells well. So, after speaking with Ryland Young, I decided to make a better plasmid.

In my construct, I placed the phage Lambda lysozyme "R gene" under constitutive control of the moderate Bla promoter from beta-lactamase. This is a promoter that appears to exhibit even expression throughout growth (reference coming, some RNA micro-array paper). Cells harboring this plasmid grow well and can be frozen if there is at least 10% glycerol present. To lyse these cells, I add 10 uL of chloroform per mL of liquid and vortex. At room temp, the cells completely lyse in about a minute. Cells harboring the lysis plasmid can be transformed with chemical methods (CsCl, TSS, etc.) but NOT by electroporation.

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